Prostaglandin ep1 receptor

ABSTRACT

(1) A novel polypeptide prostaglandin E receptor, (2) a cDNA encoding the polypeptide and a fragment selectively hybridized to the sequence of the cDNA, (3) a replication or expression vector carrying the DNA, (4) a host cell transformed with the replication or the expression vector, (5) a method for producing the polypeptide which comprises culturing the host cells, (6) a monoclonal or polyclonal antibody against the polypeptide, (7) a pharmaceutical composition containing the polypeptide or the antibody, in association with pharmaceutically acceptable excipient and/or antibody, (8) a screening method for a compound having agonistic or antagonistic activity against EP1, comprising using the polypeptide or the host cell.  
     The polypeptide of the present invention can be used for treating diseases, which is caused by over production of PGE 2 , such as pain, fever and pollakiuria.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a novel polypeptideprostaglandin E (hereinafter, simply referred to as “PGE”) receptor.

[0002] In more detail, the present invention relates to (1) a novelpolypeptide, (2) a cDNA encoding the polypeptide and a fragmentselectively hybridized to the sequence of the cDNA, (3) a replication orexpression vector carrying the DNA, (4) a host cell transformed with thereplication or the expression vector, (5) a method for producing thepolypeptide which comprises culturing the host cells, (6) a monoclonalor polyclonal antibody against the polypeptide, (7) a pharmaceuticalcomposition containing the polypeptide or the antibody, in associationwith pharmaceutically acceptable excipient and/or antibody, (8) ascreening method for a compound having agonistic or antagonisticactivity against EP1, comprising using the polypeptide, and (9) ascreening method for a compound having agonistic or antagonisticactivity against EP1, comprising using the host cells.

BACKGROUND OF THE INVENTION

[0003] Prostanoids such as prostaglandin (PG), thromboxane (TX) andleukotriene (LT) are the family of oxidized metabolites of arachidonicacid, and demonstrate various physiological actions for maintaininglocal homeostasis in the living body (The Pharmacological Basis ofTherapeutics (Gilman, A. G., Goodman, L. S., Rall, T. W., and Murad, F.,eds) 7th Ed., pp 660, Macmillan Publishing Co., New York (1985)). Theirphysiological actions are regulated through a membrane-bound receptorspecific for eachprostanoid (Annu. Rev. Pharm. Tox., 10, 213 (1989),Prostanoids and their Receptors. In Comprehensive Medicinal Chemistry.,pp 643 (1990), Pergamon Press, Oxford).

[0004] Prostaglandin E (PGE), a member of prostanoids, especiallyprostaglandin E₂ (PGE₂) participates widely in contraction andrelaxation of gastrointestinal tract, secretion of gastric acid,relaxation of smooth muscle, and release of neurotransmitters. Based onthe results obtained by the analysis of physiological andpharmacological actions of PGE2 and its site of action, it is thoughtthat there are 4 subtype receptors including EP1, EP2, EP3 and EP4(Negishi M. et al, J. Lipid Mediators Cell Signalling, 12, 379-391(1995)) and that each receptor is involved in a different signaltransduction.

[0005] Among them, it is known that EP1 is involved in pain, fever anddiuresis (Br. J. Pharmacol., 1994, 112, 735-40 European J. Pharmacol.,152 (1988) 273-279 Gen Pharmacol., September 1992, 23(5) p805-809).Therefore, it is thought that antagonizing the receptor maybe effectivefor treating pain, fever and pollakiuria. To clarify these points, it isessential to analyze a structure of EP1 receptor, a signaling and tissuedistribution of the receptor.

[0006] The amino acid sequence of EP1 receptor (Accession No.AAC37539.1)and the nucleotide sequence encoding it (Accession No.L22647) aredisclosed in Japanese Patent No. 3,090,472. However, detailed comparisonof the sequences with those of human EP1 receptor of the presentinvention revealed that there are 4 discrepancies in nucleotide sequenceand 2 in amino acid sequence, respectively.

[0007] The amino acid sequence of the polypeptide of the presentinvention was searched against the SwissProt amino acid sequencedatabases (Swiss Prot Release2.0), there was no identical sequence thatcorresponded to the polypeptide. The nucleotide sequence of the DNAencoding the polypeptide of the present invention was also searchedagainst the GenBank nucleotide sequence databases (GenBank Release70.0),no identical sequence that corresponded to the DNA was found.Accordingly, it was confirmed that the polypeptide of the presentinvention was a novel one.

DISCLOSURE OF THE INVENTION

[0008] The inventors of the present invention have successfully cloned acDNA of human EP1 receptor, expressed it in mammalian cells,demonstrated that it encoded human EP1 receptor, and thus completed thepresent invention.

[0009] Furthermore, as shown in examples bellow, the extremely highspecific binding of [³H]-PGE₂ to the receptor and the PGE₂ dependentelevation of intracellular Ca²⁺ concentration were seen in the cellsexpressed with the human EP1 receptor of the present invention. Incontrast, it has become clear that the specific binding of [³H]-PGE₂ tothe receptor but no PGE₂ dependent elevation of intracellular Ca²⁺concentration were seen in the cells expressed with the human EP1receptor which is disclosed in Japanese Patent No. 3,090,472.

[0010] The present invention relates to

[0011] (1) a polypeptide comprising the amino-acid sequence shown in SEQID NO:1,

[0012] (2) a cDNA encoding the polypeptide according to (1),

[0013] (3) a DNA according to (2), comprising the nucleotide sequenceshown in SEQ ID NO:2, or a fragment DNA selectively hybridized to thesequence,

[0014] (4) a replication or expression vector carrying the DNA accordingto (2) or (3),

[0015] (5) a host cell transformed with the replication or theexpression vector according to (4),

[0016] (6) a method for producing the polypeptide according to (1) whichcomprises culturing the host cells according to (5) under a conditioneffective to express the polypeptide according to (1),

[0017] (7) a monoclonal or polyclonal antibody against the polypeptideaccording to (1),

[0018] (8) a pharmaceutical composition containing the polypeptideaccording to (1) or the antibody according to (7), in association withpharmaceutically acceptable excipient and/or antibody,

[0019] (9) a screening method for a compound having agonistic orantagonistic activity against EP1, comprising using the polypeptideaccording to (1), and

[0020] (10) a screening method for a compound having agonistic orantagonistic activity against EP1, comprising using the host cellsaccording to (5).

DETAILED DESCRIPTION OF THE INVENTION

[0021] A DNA selectively hybridizing to the nucleotide sequence shown inSEQ ID NO:2 is generally at least 70%, preferably at least 80 or 90% andmore preferably at least 95% homologous to the DNA over a region of atleast 100, preferably at least 150, for example 200, 250 or 300contiguous nucleotides. Such a DNA homologue is referred to as a DNA ofthe present invention.

[0022] The selectively hybridizing DNAs contain complementary sequencesagainst above sequence. The hybridization on stringent condition ispreferred.

[0023] Generally, a fragment of DNA comprising nucleotide sequence shownin SEQ ID NO:2 is at least 10, preferably at least 15, for example 20,25, 30 or 40 nucleotides in length, and is also referred to as a DNA ofthe present invention.

[0024] The DNA of the present invention can be obtained by recombinantDNA technologies, chemical synthesis, or methods known by a man skilledin the art.

[0025] A further embodiment of the present invention providesreplication or expression vectors carrying the DNA of the presentinvention. The vectors may be, for example, plasmid, virus or phagevectors provided with an origin of replication, optionally a promoterfor the expression of the DNA and a regulator of the promoter. Thevector may contain one or more selectable marker genes, for example anampicillin resistance gene.

[0026] A further embodiment of the present invention provides host cellstransformed with the replication or expression vector carrying the DNAof the present invention, including the DNA of which nucleotide sequenceis shown in SEQ ID NO:2. The cells can be for example bacterial, yeast,insect or mammalian cells.

[0027] A further embodiment of the present invention provides a methodfor producing the polypeptide, which comprises culturing host cells ofthe present invention, under conditions effective to express thepolypeptide of the present invention.

[0028] As a polypeptide of the present invention, those which havedeficiency in a part of the amino acid sequence shown in SEQ ID NO:1(e.g., a polypeptide comprised of the only essential sequence forrevealing a biological activity in their mature amino acid sequence),those which have a part of their amino acid sequence replaced by otheramino acids (e.g., those replaced by an amino acid having a similarproperty) and those which have other amino acids added or inserted intoa part of their amino acid sequence, as well as those comprising theamino acid sequence shown in SEQ ID NO:1, are provided.

[0029] As known well, there are one to six kinds of codon encoding oneamino acid (for example, one kind of codon for Methionine (Met), and sixkinds of codon for leucine (Leu) are known). Accordingly, the nucleotidesequence of a DNA can be changed without changing the amino acidsequence of a polypeptide.

[0030] The DNA of the present invention according to (2) includes everygroup of nucleotide sequences encoding polypeptide shown in SEQ ID NO:1.There is a probability that yield of a polypeptide is improved bychanging a nucleotide sequence.

[0031] The DNA specified in SEQ ID NO:2 according to (3) is oneembodiment of the DNA according to (2), and indicates the sequence ofnatural form.

[0032] The DNA having the nucleotide sequence shown in SEQ ID NO:2 isprepared by the following methods:

[0033] That is:

[0034] (i) The mRNA is prepared from cells that produce the polypeptideof the present invention,

[0035] (ii) The first-strand DNA (a single-strand DNA) is synthesized byusing the mRNA as a template, then the second-strand DNA (adouble-strand DNA) is synthesized (preparation of DNA),

[0036] (iii) The DNA fragment is inserted into an appropriate plasmidvector,

[0037] (iv) Host cells are transformed with the recombinant vectors(preparation of cDNA library),

[0038] (v) The plasmid containing the aimed DNA is isolated from thecDNA library by using hybridization method, and

[0039] (vi) The aimed DNA is prepared by sequencing the DNA insert ofthe plasmid.

[0040] Detailed description of each step is as follows:

[0041] In step (i), mRNA is isolated from human organ that expresses EP1receptor, preferably from tissue or cell line such as brain, placenta,neuroblastoma (T98G etc.), and erythroleukemia (Hel etc.), by the methodof Okayama, H et al. (Method in Enzymology 154, 3 (1987)) or Chirgwin,J. M. et al. (Biochem. 18, 5294 (1979)).

[0042] In steps (ii), (iii) and (iv), a cDNA library is prepared by themodified method of Gubler & Hoffman (Gene 25, 263 (1983)). For plasmidvector to be used in step (iii), various vectors, which is functional inE. coli (for example pBR322) or in B. subtilis (for example pUB110), areknown. Preferably λ-ZAPII that is functional in E. coli is used. In step(iv), a host cell can be chosen from a variety of host cells known,preferably competent cells (prepared by the method described in Gene 96,23 (1990)) of E. coli strain DH5 is used.

[0043] Recently, cDNA libraries prepared from a variety of tissues ofanimals, which are already on the market, can also be used preferably.

[0044] In step (v), hybridization is carried out by the known methodssuch as plaque hybridization or colony hybridization (Gene 10, 63(1980)) etc. As a probe, a DNA of EP1 receptor from another animal thanhuman, a fragment thereof, or a DNA homologous to said DNA could beused.

[0045] In step (vi), sequencing can be performed by the known methodsuch as the method of dideoxy terminator or the method of Maxam-Gilbert.

[0046] Once the nucleotide sequence shown in SEQ ID NO:2 is identified,then the DNA of the present invention can be obtained by chemicalsynthesis, PCR method, or hybridization method by using the fragment ofsaid nucleotide sequence as a probe. Furthermore, a necessary amount ofthe DNA of the present invention can be obtained by introducing a vectorcontaining the DNA to an appropriate host cell, followed byproliferating the cells.

[0047] The polypeptides of the present invention (SEQ ID NO:1) may beprepared by:

[0048] (1) isolating and purifying from an organism or a cultured cell,

[0049] (2) chemically synthesizing, or

[0050] (3) using recombinant DNA technology, preferably, by the methoddescribed in (3) in an industrial production.

[0051] Examples of expression system (host-vector system) for producinga polypeptide by using recombinant DNA technology are the expressionsystems of bacteria, yeast, insect cells and mammalian cells.

[0052] In the expression of the polypeptide, for example, in E. Coli,the expression vector is prepared by connecting the DNA encoding matureprotein (for example the DNA shown in SEQ ID NO:2) to the downstream ofa proper promoter (e.g., trp promoter, lac promoter, λ PL promoter, andT7 promoter), and then inserting it into a vector (e.g., pBR322, pUC18and pUC19) which functions in an E. coli strain. Then, an E. coli strain(e.g., E. coli strain DH1, E. coli strain JM109 and E. coli strainHB101) which is transformed with the expression vector described abovemay be cultured in an appropriate medium to obtain the desiredpolypeptide. When a signal peptide of bacteria (e.g., signal peptide ofpel B) is utilized, the desired polypeptide may be also released inperiplasm. Furthermore, a fusion protein with other polypeptide may bealso produced easily.

[0053] In the expression of the polypeptide, for example, in mammaliancells, for example, the expression vector is prepared by inserting theDNA encoding the nucleotide sequence shown in SEQ ID NO:2 into thedownstream of a proper promoter (e.g., SV40 promoter, LTR promoter andmetallothionein promoter) in a proper vector (e.g., vaccinia virusvector and SV40 vector). A proper mammalian cell (e.g., monkey COS-7cell, Chinese hamster CHO cell, mouse L cell etc.) is transfected withthe expression vector thus obtained, and then the transfectant iscultured in a proper medium, the aimed polypeptide can be secreted intothe culture medium. The polypeptides thus obtained can be isolated andpurified by conventional biochemical methods.

[0054] The present invention also provides monoclonal or polyclonalantibodies against the polypeptide of the present invention. The presentinvention further provides a process for production of monoclonal orpolyclonal antibodies against the polypeptide of the present invention.Monoclonal antibodies can be prepared by common hybridoma technologyusing the polypeptide of the present invention or fragments thereof asan immunogen. Polyclonal antibodies can also be prepared by common meanswhich comprises inoculating host animals (for example rat or rabbit)with the polypeptide of the present invention and recovering the immuneserum.

[0055] The present invention also provides pharmaceutical compositionscontaining a polypeptide of the present invention, or an antibodythereof, in association with a pharmaceutically acceptable excipientand/or carrier.

[0056] The DNA of the present invention may also be inserted into thevectors described above in an antisense orientation in order to produceantisense RNA. Such antisense RNA may be used in controlling the levelof the polypeptide of the present invention in a cell.

Administration and Dosing

[0057] For treating diseases such as pain, fever and pollakiuria,administration of the polypeptide of the present invention or itsantibodies can be carried out in systemic or local, generally peroral orparenteral ways. Oral, intravenous and intracerebroventricularadministrations are preferred.

[0058] The dosage to be administered depends upon age, body weight,symptom, desired therapeutic effect, route of administration, andduration of the treatment etc. In human adults, one dose per person isgenerally between 100 μg and 100 mg by oral administration up to severaltimes per day, and between 10 μg and 100 mg by parenteral administrationup to several times per day.

[0059] As mentioned above, the doses to be used depend upon variousconditions. Therefore, there are cases in which doses lower than orgreater than the ranges specified above may be used.

[0060] The compounds of the present invention may be administered assolid compositions, liquid compositions or other compositions for oraladministration, as injections, liniments or suppositories etc. forparenteral administration.

[0061] Examples of solid compositions for oral administration includecompressed tablets, pills, capsules, dispersible powders and granules.Examples of capsules include soft capsules and hard ones.

[0062] In such compositions, one or more of the active compound(s) is orare admixed with at least one inert diluent (such as lactose, mannitol,glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch,polyvinylpyrrolidone, magnesium metasilicate aluminate, etc.). Thecompositions may also comprise, as is normal practice, additionalsubstances other than inert diluents: e.g. lubricating agents (such asmagnesium stearate etc.), disintegrating agents (such as cellulosecalcium glycolate, etc.), stabilizing agents (such as human serumalbumin, lactose etc.), and assisting agents for dissolving (such asarginine, asparaginic acid etc.).

[0063] The tablets or pills may, if desired, be coated with a film ofgastric or enteric materials such as sugar, gelatin, hydroxypropylcellulose or hydroxypropylmethyl cellulose phthalate, or be coated withmore than two films. And then, coating may include containment withincapsules of absorbable materials such as gelatin.

[0064] Liquid compositions for oral administration may containpharmaceutically acceptable emulsions, solutions, suspensions, syrupsand elixirs, and also may contain inert diluent(s) commonly used(purified water, ethanol etc.). Besides inert diluents, suchcompositions may also comprise adjuvants (such as wetting agents,suspending agents, etc.), sweetening agents, flavoring agents, perfumingagents, and preserving agents.

[0065] Other compositions for oral administration include spraycompositions which may be prepared by known methods and which compriseone or more of the active substance(s). Spray compositions may compriseadditional substances other than inert diluents: e.g. stabilizing agents(sodium sulfite etc.), isotonic buffer (sodium chloride, sodium citrate,citric acid, etc.). For preparation of such spray compositions, forexample, the method is described in the U.S. Pat. Nos. 2,868,691 and3,095,355 (herein incorporated in their entireties by reference) may beused.

[0066] Injections for parenteral administration include sterile aqueousor non-aqueous solutions, suspensions and emulsions. In suchcompositions, one or more active compound(s) is or are admixed with atleast one inert aqueous diluent(s) (distilled water for injection,physiological salt solution, etc.) or inert non-aqueousdiluents(s)(propylene glycol, polyethylene glycol, olive oil, ethanol,POLYSOLBATE 80™, etc.).

[0067] Injections may comprise additional compound other than inertdiluents: e.g. preserving agents, wetting agents, emulsifying agents,dispersing agents, stabilizing agent (such as human serum albumin,lactose, etc.), and assisting agents such as assisting agents fordissolving (arginine, asparaginic acid, etc.).

INDUSTRIAL APPLICABILITY

[0068] Since the polypeptide of the present invention specifically boundPGE₂, the polypeptide can be used for treating diseases, which is causedby over production of PGE₂, such as pain, fever and pollakiuria. Thepolypeptide may also be used for screening substances having agonisticor antagonistic activities against EP1 receptor.

[0069] Quantitative analysis of the polypeptide of the present inventionin the body can be performed using polyclonal or monoclonal antibodiesagainst the polypeptide, and thus can be used in studies on relationshipbetween the polypeptide and diseases, or diagnosis of diseases, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

[0070]FIG. 1 shows a scatchard blot in binding assays using cellsexpressing human EP1 receptor of the present invention.

[0071]FIG. 2 shows a scatchard blot in binding assays using cellsexpressing human EP1 receptor described in Japanese Patent No.3,090,472.

[0072]FIG. 3 shows changes of intracellular Ca²⁺ concentration in theexperiments using cells expressing human EP1 receptor of the presentinvention or that of Japanese Patent No. 3,090,472.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0073] The invention is illustrated by the following examples, but notlimit the invention.

EXAMPLE 1 cDNA Cloning of Human EP1 Receptor

[0074] Erythroleukemia cell line, HEL cells were cultured and mRNA wasprepared from the cells by the conventional method. RT-PCR (reversetranscrived polymerase chain reaction) was carried out in two steps withthe mRNA thus obtained. After the synthesis of first strand DNA byreverse transcription, the following reaction mixture was prepared.

[0075] Reaction mixture: HEL cDNAs (1 μl), primer 1 (0.5 μM), primer 2(0.5 μM), PCR buffer (Tris-HCl (pH 8.3, 10 mM), KCl (50 mM), MgCl2 (1.5mM)), dNTPs (mixtures of each 0.25 mM of DATP, dCTP, dGTP and dTTP), Taqpolymerase (0.5 unit/μl) and 10% DMSO. Primer 1: 5′-TGGCGCCTGACATGAGCCCT T-3′ (SEQ ID NO:3) Primer 2: 5′-TCGTTGGGCC TCTGGTTGTG CTTA-3′(SEQ ID NO:4)

[0076] The conditions of PCR were as follows;

[0077] First step: [(94° C., 1 min.), (62° C., 2 min.), and (72° C., 3min.)]×25 cycles,

[0078] Second step: [(94° C., 1 min.), (55° C., 2 min.), and (72° C., 3min.)]×25 cycles.

[0079] The PCR product was subjected to agarose gel electrophoresis, the1250-bp band having the aimed length was obtained. The band waspurified, inserted into pT7 Bleu T-Vector (Novagen) (hereinafter, simplyreferred to as “pT7 Bleu hEP1”), and sequenced by cycle-sequencingmethod using fluorescence-dye terminator of ABI (Applied BiosystemsInc.) which is based upon the di-deoxynucleotide chain terminationmethod of Sanger F. et al. DNA sequencer purchased from ABI was used forreading the nucleotide sequence. The nucleotide sequence of the cDNAencoding hEP1 receptor was shown in SEQ ID NO:2, and the deduced aminoacid sequence in SEQ ID NO:1.

[0080] The nucleotide sequence of the hEP1 receptor of the presentinvention and the predicted amino acid sequence thereof were comparedwith those of the hEP1 receptor described in Japanese Patent No.3,090,472 (hereinafter, a number written on right shoulder of anucleotide or an amino acid shows a position from “A” of ATG,translation initiation codon, and a position from “Met” that istranslation initiation amino acid, respectively).

[0081] G²¹¹ in the nucleotide sequence of the present invention wassubstituted by “A” in that of Japanese Patent No. 3,090,472.Hereinafter, it is shown as “G²¹¹→A”. Similar substitutions were alsofound in T⁶⁸⁹→A, A⁶⁹⁰→T and A⁹⁹⁹→G, thus there were 4 differences in thenucleotide sequences between them. Based upon these differences, it wasfound that there were 2 differences in amino acid sequences betweenthem, i.e., Ala⁷¹→Thr and Leu²³⁰→His.

EXAMPLE 2 Binding Assays with the Cells Expressing Human EP1 Receprtor

[0082] 1) Gene Transfection Into CHO Cells and Isolation of ClonesExpressing Human EP1 Receptor

[0083] The cDNA insert was cut off from pT7BluehEP1 by EcoR1 digestion,and inserted into EcoR1 site of expression vector pdKCR-dhfr in theproper orientation. Approximately 25-50 ug of expression plasmid wasdigested completely with proper restriction enzymes (SalI, SacII etc.),purified by Phenol/CIAA extraction, CIAA extraction and ethanolprecipitation, and dissolved in 50 μl of sterilized water. CHO-dhfr(−)cells used as host were suspended in 1.0-1.5×10⁶ cells/0.8 ml of PBSucbuffer (sucrose 272 mM, K⁺PO₄ ²⁻ buffer (7 mM, pH 7.4) and MgCl₂ (1mM)). Ten microliter of Plasmid DNA and 0.8 ml of cell suspension wereadded into Gene Pulser Cuvette (Bio-rad, Cat. No.165-2088), incubatedfor 10 minutes on ice, and pulsed with 25 uF/200-500V using Gene Pulser(Bio-rad). After additional 10 minutes incubation on ice, cells weresuspended in 10 ml of medium (permissive medium; α-MEM (DainipponPharmaceutical Co., Ltd.) containing 100×HT supplement (GIBCO BRL),penicillin (100 U/ml), streptomycin (100 ug/ml) and 10% dialyzed FCS(GIBCO)), transferred into culture flask (75 cm²), and cultured forapproximately 48 hours in CO₂ incubator. The cells were harvested aftertrypsin treatment, suspended in selection medium (nonpermissive medium;α-MEM (Dainippon Pharmaceutical Co., Ltd.) containing penicillin (100U/ml), streptomycin (100 ug/ml) and 10% dialyzed FCS (GIBCO)), andcultured.

[0084] To isolate a single colony, after several days to one-weekcultivation, when colonies (20-30 cells/colony) were grown well,colonies were isolated as follows. The culture medium was removed fromflasks, and cells were washed with PBS. A filter paper, which was cutinto approximately 5 mm×5 mm, autoclaved and soaked in trypsin solution,was added onto each colony and incubated at 37° C. for 3 minutes. Eachcolony was removed from the flask with the filter paper, transferredinto 24-well plate, and 1 ml/well of selection medium was added. Afteradditional cultivation, the promising clones were selected based on aform, multiplication ability, etc. of a cell.

[0085] Alternatively, the limited dilution method was carried out. Afterapproximately one-week cultivation, in due course when cells weregrowing well, cells were harvested and inoculated into several 96-wellplates at 2-5 cells/well. The promising clones were selected based on aform, multiplication ability, etc. of a cell, and transferred into24-well plate. 2) Preparation of membrane fraction

[0086] The CHO cloned cells expressing human EP1 receptor obtained bythe above method were cultured and membrane fraction was prepared by thefollowing methods.

[0087] The cells were cultured in large scale using 500 cm² trey(Sumitomo Bakelite Co., Ltd.) with α-MEM (Dainippon Pharmaceutical Co.,Ltd.) containing penicilin (100 U/ml), streptmycin (100 ug/ml) and 10%dialyzed FCS (GIBCO). The cells were washed with PBS(−), recovered usingcell-scraper, centrifuged at 1,000 rpm for 3 minutes, and the cellpellets were stored at −80° C.

[0088] The following operations for preparing cell membranes wereperformed at 4° C. or below. The cell pellets, which were harvested from50-150 treys, were added with 10-fold volume of buffer A cooled on ice,and homogenized using Potter homogenizer (1,000 rpm, 5 strokes). Aftercentrifugation at 1,000×g for 10 minutes, the lysate was centrifugedfurther at 100,000×g for 15 minutes. The pellets thus obtained weresuspended in buffer B and stored at −80° C. as a membrane fraction untiluse.

[0089] Buffer A:

[0090] Tris-HCl (20 mM, pH 7.5) containing EDTA (1 mM), MgCl₂ (10 mM),PMSF (0.1 mM), pepstatin A (10 μM) and indomethacin (20 μM);

[0091] Buffer B:

[0092] KPi (pH 6.0, 10 mM) containing EDTA (1 mM), MgCl₂ (10 mM) andNaCl (0.1M).

[0093] 3) Binding Assays

[0094] Reaction mixtures (200 μl) containing the membrane fraction,which was prepared by the above method and contains human EP1 receptor,and various concentrations of [3H]-PGE₂ were incubated at roomtemperature for a certain time, separated by rapid filtration using CellHarvester (Brandel). The membrane fraction was recovered on glass filterand washed several times with the buffer cooled on ice (1-2 ml/wash).Residual [3H]-PGE₂ bound to the filter (total binding) was quantitatedby liquid scintillation counter. Nonspecific binding was quantitated ina similar manner in the presence of 10 μM of PGE₂. Specific binding wasdefined as the difference between total binding and nonspecific binding.

[0095] The specific binding thus obtained was analyzed by Scatchardplots, an equilibrium dissociation constant (Kd; nM) and a maximumnumber of specific binding sites (Bmax; fmol/mg protein) weredetermined. The result obtained using the cells expressing human EP1 ofthe present invention was shown in FIG. 1, and the result obtained usingthe cells expressing human EP1 described in Japanese Patent No.3,090,472 in FIG. 2, respectively. The amount of hEP1 mRNA in the cellsexpressing human EP1 receptor used in FIGS. 1 and 2 was analyzed byquantitative PCR, no difference was found, suggesting the human EP1receptor were expressed on both cells in equal level.

EXAMPLE 3 Measurement of Intracellular Ca²⁺ Concentration in CellsExpressing Human EP1 Receptor

[0096] The cells were cultured in 500 cm² treys to reach confluence,washed with PBS(−), and peeled off by pipeting with PBS(−) on ice. Aftercentrifugation at 800 rpm for 3 minutes, the cell pellet was suspendedin medium A (15 ml) and incubated at 37° C. for 50 minutes followed byat room temperature for 10 minutes. After re-centrifugation the pelletwas suspended in medium B (1-2×10⁶/ml) and used for experiments. In thecase that cell suspension contains Fura2-AM (Dojin), the operations wereshielded the light by aluminum foil. Stirring the cell suspensions (400μl) at 600 rpm, the changes of intracellular Ca²⁺ concentration weremeasured by monitoring the changes of fluorescence intensity at both 340nm and 380 nm and the ratio thereof, using CAM-220 Spectrofluorometer(Nihon Bunko Kogyo).

[0097] Medium A:

[0098] α-MEM containing fura2-AM (5 μM), indomethacin (10 μM),HEPES-NaOH (pH 7.4, 10 mM) and 10% FCS, Medium B:

[0099] α-MEM containing indomethacin (1 μM), HEPES-NaOH (pH 7.4, 10 mM)and 0.1% BSA.

[0100] The changes in intracellular Ca²⁺ concentration using the cellsexpressing human EP1 receptor of the present invention and the cellsexpressing human EP1 receptor described in Japanese Patent No.3,090,472were shown in FIG. 3. In FIG. 3, “reference” means the result obtainedby using the cells expressing human EP1 receptor described in JapanesePatent No. 3,090,472.

[0101] As obvious from examples 2 and 3, the extremely high specificbinding of [³H]-PGE₂ to the receptor and the PGE₂ dependent elevation ofintracellular Ca²⁺ concentration were detected in the cells expressinghuman EP1 receptor of the present invention.

[0102] In contrast, the specific binding of [³H]-PGE₂ to the receptorwas detected, however, the PGE₂ dependent elevation of intracellularCa²⁺ concentration was not detected at all in the cells expressing humanEP1 receptor described in Japanese Patent No. 3,090,472.

1 4 1 402 PRT Homo sapien 1 Met Ser Pro Cys Gly Pro Leu Asn Leu Ser LeuAla Gly Glu Ala Thr 1 5 10 15 Thr Cys Ala Ala Pro Trp Val Pro Asn ThrSer Ala Val Pro Pro Ser 20 25 30 Gly Ala Ser Pro Ala Leu Pro Ile Phe SerMet Thr Leu Gly Ala Val 35 40 45 Ser Asn Leu Leu Ala Leu Ala Leu Leu AlaGln Ala Ala Gly Arg Leu 50 55 60 Arg Arg Arg Arg Ser Ala Ala Thr Phe LeuLeu Phe Val Ala Ser Leu 65 70 75 80 Leu Ala Thr Asp Leu Ala Gly His ValIle Pro Gly Ala Leu Val Leu 85 90 95 Arg Leu Tyr Thr Ala Gly Arg Ala ProAla Gly Gly Ala Cys His Phe 100 105 110 Leu Gly Gly Cys Met Val Phe PheGly Leu Cys Pro Leu Leu Leu Gly 115 120 125 Cys Gly Met Ala Val Glu ArgCys Val Gly Val Thr Arg Pro Leu Leu 130 135 140 His Ala Ala Arg Val SerVal Ala Arg Ala Arg Leu Ala Leu Ala Ala 145 150 155 160 Val Ala Ala ValAla Leu Ala Val Ala Leu Leu Pro Leu Ala Arg Val 165 170 175 Gly Arg TyrGlu Leu Gln Tyr Pro Gly Thr Trp Cys Phe Ile Gly Leu 180 185 190 Gly ProPro Gly Gly Trp Arg Gln Ala Leu Leu Ala Gly Leu Phe Ala 195 200 205 SerLeu Gly Leu Val Ala Leu Leu Ala Ala Leu Val Cys Asn Thr Leu 210 215 220Ser Gly Leu Ala Leu Leu Arg Ala Arg Trp Arg Arg Arg Ser Arg Arg 225 230235 240 Pro Pro Pro Ala Ser Gly Pro Asp Ser Arg Arg Arg Trp Gly Ala His245 250 255 Gly Pro Arg Ser Ala Ser Ala Ser Ser Ala Ser Ser Ile Ala SerAla 260 265 270 Ser Thr Phe Phe Gly Gly Ser Arg Ser Ser Gly Ser Ala ArgArg Ala 275 280 285 Arg Ala His Asp Val Glu Met Val Gly Gln Leu Val GlyIle Met Val 290 295 300 Val Ser Cys Ile Cys Trp Ser Pro Met Leu Val LeuVal Ala Leu Ala 305 310 315 320 Val Gly Gly Trp Ser Ser Thr Ser Leu GlnArg Pro Leu Phe Leu Ala 325 330 335 Val Arg Leu Ala Ser Trp Asn Gln IleLeu Asp Pro Trp Val Tyr Ile 340 345 350 Leu Leu Arg Gln Ala Val Leu ArgGln Leu Leu Arg Leu Leu Pro Pro 355 360 365 Arg Ala Gly Ala Lys Gly GlyPro Ala Gly Leu Gly Leu Thr Pro Ser 370 375 380 Ala Trp Glu Ala Ser SerLeu Arg Ser Ser Arg His Ser Gly Leu Ser 385 390 395 400 His Phe 2 1209DNA Homo sapien protein_bind (1)..(1209) 2 atgagccctt gcgggcccctcaacctgagc ctggcgggcg aggcgaccac atgcgcggcg 60 ccctgggtcc ccaacacgtcggccgtgccg ccgtcgggcg cttcgcccgc gctgcccatc 120 ttctccatga cgctgggcgccgtgtccaac ctgctggcgc tggcgctgct ggcgcaggcc 180 gcgggccgcc tgcgacgccgccgctcggcc gccaccttcc tgctgttcgt ggccagcctg 240 ctggccaccg acctggcgggccacgtgatc ccgggcgcgc tggtgctgcg tctgtacact 300 gcggggcgcg ctccggccggcggggcctgc cacttcctgg gcggctgcat ggtcttcttc 360 ggcctgtgcc cgctgctgctgggctgtggc atggccgtgg agcgctgcgt gggcgtcacg 420 cggccgctgc tccacgccgcgcgggtctcg gtcgcccgcg cgcgcctggc gctggccgcg 480 gtggccgcgg tggccttggccgtggcgctg ctgccgctgg cgcgcgtggg ccgctatgag 540 ctgcagtacc cgggcacgtggtgcttcatc ggcctgggtc ccccgggcgg ctggcgccag 600 gcactgcttg ctggcctcttcgccagcctc ggcctggtcg cgctcctcgc cgcgctggtg 660 tgcaacacgc tcagcggcctggccctgcta cgcgcccgct ggcgacgccg ctcccgacgg 720 cctcccccgg cctcaggccccgacagccgg cgtcgctggg gggcgcacgg accccgctcg 780 gcctccgcct cgtccgcctcgtccatcgct tcggcctcca ccttctttgg cggctctcgg 840 agcagcggct cggcacgcagagctcgcgcc cacgacgtgg agatggtggg ccagcttgtc 900 ggtatcatgg tggtgtcgtgcatctgctgg agcccaatgc tggtgttggt ggcgctggcc 960 gtcggcggct ggagctctacctccctgcag cggccactat tcctggccgt gcgccttgcc 1020 tcctggaacc agatcctggacccttgggtg tacatcctac tgcgccaggc cgtgctgcgc 1080 caactgcttc gcctcttgcccccgagggcc ggagccaagg gcggccccgc ggggctgggc 1140 ctaacaccga gcgcctgggaggccagctcg ctgcgcagct cccggcacag cggcctcagc 1200 cacttctaa 1209 3 21 DNAArtificial Sequence Forward primer 3 tggcgcctga catgagccct t 21 4 24 DNAArtificial Sequence Reverse primer 4 tcgttgggcc tctggttgtg ctta 24

1. A polypeptide comprising the amino acid sequence shown in SEQ IDNO:1.
 2. A cDNA encoding the polypeptide according to claim
 1. 3. A DNAaccording to claim 2, comprising the nucleotide sequence shown in SEQ IDNO:2, or a fragment DNA selectively hybridized to the sequence.
 4. Areplication or expression vector carrying the DNA according to claims 2or
 3. 5. A host cell transformed with the replication or expressionvector according to claim
 4. 6. A method for producing the polypeptideaccording to claim 1 which comprises culturing the host cell accordingto claim 5 under a condition effective to express the polypeptideaccording to claim
 1. 7. A monoclonal or polyclonal antibody against thepolypeptide according to claim
 1. 8. A pharmaceutical compositioncontaining the polypeptide according to claim 1 or the antibodyaccording to claim 7, in association with pharmaceutically acceptableexcipient and/or antibody.
 9. A method for screening a compound havingagonistic or antagonistic activity against EP1, comprising using thepolypeptide according to claim
 1. 10. A method for screening a compoundhaving agonistic or antagonistic activity against EP1, comprising usingthe host cell according to claim 5.